About 20 million Americans have kidney disease. The number of people diagnosed with kidney disease has doubled each decade for the last two decades impacting on human suffering and enormous Medicare costs imposed by end-stage renal failure. Renal ultrafiltration is located within the renal glomerulus and performed by highly specialized podocyte cells. Podocyte foot processes (FPs) and the interposed slit diaphragms (SDs) cover the outer aspect of the glomerular filtration barrier and form a final barrier to protein loss. Damage of podocytes results in proteinuria and may lead to progressive decline of renal function. It is therefore in the interest of the public health to define the regulation of podocyte structure and function at the cellular and molecular levels and identify molecular targets involved in early structural changes leading to podocyte damage and the development of proteinuria. We have made the novel finding that cathepsin L enzyme is present within the cytoplasm of podocytes during nephrotic syndrome and cleaves CD2AP, an important SD protein. Cathepsin L is a lysosomal protease that has a broad biological significance such as intracellular protein degradation, activation of enzyme precursors, and tumor invasion. Normally located in lysosomes, cathepsin L can be found in the cytoplasm or can be secreted under certain conditions. We have developed a novel murine model of transient nephrotic syndrome that shares key features with human nephrotic syndrome which allows us to delineate proteolytic processes in podocytes resulting from the induction of cathepsin L. We propose 3 Specific Aims to unravel the function of extralysosomal cathepsin L expression and activity in podocytes. In the first Specific Aim, we propose to analyze the course and severity of proteinuria in mice lacking cathepsin L and how the lack of this enzyme affects CD2AP binding interactions with other SD proteins. We will then test the hypothesis in detail that the SD protein CD2AP is a proteolytic target protein of cathepsin L and explore the cell-junction stability of cathepsin L cleavage resistant CD2AP mutants (Specific Aim 2). In Specifc Aim 3, we will address the biological significance of cleaved CD2AP fragments and analyse the effects of such peptides on the actin based lysosomal degradome using organellar proteomics. The proposed role of proteolytic processing of podocyte structural and regulatory proteins during nephrotic syndrome represents a novel concept in the molecular work-up of proteinuria. If our hypothesis is correct, our work will have broad significance for the basic understanding of glomerular pathology e.g. the mechanism of podocyte FP effacement. Uncovering the role of podocyte proteolysis will help to develop novel pharmaco-therapeutics (such as cathepsin L resistant CD2AP mutants or small molecules blocking CD2AP cleavage) to tackle proteinuria and progression of glomerular disease.